1. Field of the Invention
This invention relates to the field of acetone detection. Acetone-specific enzyme systems and sensors capable of qualitatively and/or quantitatively detecting acetone have now been developed. These enzyme systems can be incorporated into relatively inexpensive, simple and/or portable enzyme-based sensors particularly suited for detecting acetone in environmental or biological samples, for example, mammalian breath samples.
2. Description of Related Art
Acetone Sources
Acetone may be detected in liquids and gases present in or obtained from biological organisms and various environments. For example, acetone may be detected in environments such as: natural environments, including soils, sediments, streams, or wetlands; indoor and outdoor work and home environments; and waste environments, including waste storage ponds and waste disposal sites. Acetone may be found in environmental and biological liquids and gases due to introduction of acetone to the environment or organism from an external source. Thus, environmental acetone may result from leakage, leaching, waste discharge, or solvent evaporation, or from emission of combustion gases released by burning wood or plastic or by operating petrochemical internal combustion engines. Likewise, in a living organism, acetone may be present due to ingestion, inhalation, or absorption from an external source.
Acetone may also be found in such environmental and biological liquids and gases due to internal generation of acetone by the environment tested (whether by chemical reaction or by biological production) or by the organism tested (for example, microbes, animals, etc.). Thus, the published literature reports that, acetone:                occurs as a biodegradation product of sewage, solid wastes and alcohols, and as an oxidation product of humic substances. Acetone has been detected in a variety of plants and foods including onions, grapes, cauliflower, tomatoes, morning glory, wild mustard, milk, beans, peas, cheese and chicken breast. Natural emissions from a variety of tree species contain acetone vapour.        
J D Reisman, Environmental Health Criteria for Acetone (Draft), Environmental Health Criteria No. 207, International Programme on Chemical Safety (INCHEM) (1998). For example, acetone may form chemically within an environment by atmospheric oxidation of plant terpenes (Fruekilde et al. (1998)).
Living organisms may internally generate acetone via a number of enzymatic routes. In microbes, acetone may be synthesized, for example: by oxidation of isopropanol, as may be performed by Mycobacterium spp., including M. vaccae; by desulfonation of 2-propanesulfonate, as may be performed by Rhodococcus spp. and Comamonas spp., including C. acidovorans; and by decarboxylation of acetoacetate, as may be performed by Clostridium spp., including C. acetobutylicum, C. butyricum, and C. saccharoperhutylacetonium. 
In the vertebrate animals, including humans, the most common route of acetone synthesis is by ketone body formation. Ketone bodies are compounds produced from the oxidation of lipids by the liver and used as an energy source when glucose is not readily available. The main compounds classified as ketone bodies include acetoacetic acid, β-hydroxybutyric acid, and acetone. Ketones are always present in the body, and their levels increase during fasting and prolonged exercise. Oxidation of fatty acids in liver mitochondria produces acetyl-coenzyme A, which can be further oxidized via the citric acid cycle or undergo a process called ketogenesis. Ketogenesis occurs primarily when glucose is not available as an energy source and converts acetyl-coenzyme A to acetoacetate or β-hydroxybutyrate. The liver releases acetoacetate and β-hydroxybutyrate to the bloodstream where it is carried to peripheral tissues and is used as an alternative energy source. Acetoacetate is a β-ketoacid and slowly undergoes spontaneous non-enzymatic decarboxylation to acetone and CO2 (Scheme 1).

In tetrapod vertebrates, including mammals, the acetone thus formed is detectable in respiration as a result of blood gas exchange in the lung.
Breath acetone levels have been correlated with blood acetone levels and so may be used as an accurate indicator of blood acetone content. Thus, elevated breath acetone levels have been demonstrated, in clinical studies of otherwise healthy human subjects, to be a reliable indicator of fat metabolism and projected weight loss. Acetone is present in human breath at endogenous levels of about 0.2-0.5 ppm (v/v) and increases to and above 5-25 ppm for otherwise healthy individuals on long term, low carbohydrate diets. Similarly, breath acetone concentrations increase in individuals on a high-fat diet. Each of these dietary conditions is called a “benign dietary ketosis.” Breath acetone concentration likewise increases during short-term fasting, a condition known as “fasting ketosis,” and after prolonged exercise, a condition known as “post-exercise ketosis.” Under starvation conditions (or long-term fasting) or in diabetics whose insulin levels drop too low, the concentration of breath acetone may become abnormally high (up to 70 ppm or higher), indicating conditions called, respectively, “metabolic ketoacidosis” or “diabetic ketoacidosis,” diabetic ketoacidosis being a potentially fatal condition. In each of these conditions, elevated acetone levels can be detected in the breath of juveniles or adults.
In addition to acetone elevation by benign dietary, fasting, and post-exercise ketosis, and by diabetic and metabolic ketoacidosis, other conditions and diseases can also generate elevated blood acetone, and thereby elevated breath acetone, levels. For example, blood acetone elevation is also observed in, for example: 1) the female reproductive cycle (for example, during pregnancy or during the post-partum interval preceding resumption of ovulation); 2) the neonatal stage of development; 3) hypoglycemia (for example, hypoglycemia of childhood or hypoglycemia caused by eating disorders or prolonged vomiting); 4) inborn metabolic diseases (for example, maple syrup urine disease); 5) liver dysfunction (for example, end-stage liver disease or hepatic ischemia); 6) glucocorticoid deficiency; 7) growth hormone deficiency; 8) acute pancreatitis resulting from viral infection (for example, systemic cytomegalovirus infection); 9) treatment with nucleoside analogs (for example, in anti-retroviral therapy for HIV); 10) isopropanol ingestion or intoxication; 11) ethanol intoxication; and 12) salicylate intoxication. In these conditions, too, acetone can be detected by, for example, breath analysis of children or adults.
Thus, detection of acetone can be useful in a number of medically important applications. For example, medical reports have identified obesity as a primary risk factor in diabetes, hypertension, coronary heart disease, hypercholesterolemia and stroke. In many cases of obesity, a controlled weight loss program can reverse these serious life-threatening diseases. Acetone is a metabolite that can be detected to monitor the progress in and compliance with such a weight loss program. Similarly, detection of acetone can be used to alert diabetic subjects to the onset of ketoacidosis or to obtain a preliminary indication of the need to diagnose a subject for any of the other medical conditions or diseases in which elevated acetone may be found. Therefore, acetone is a key diagnostic metabolite that can be used as a means to monitor diet compliance, weight loss progress, medical treatment regimen compliance, diabetes, and health wellness in subjects of all ages.
The Field of Acetone Detection
Acetone can be detected in any of the above-described situations, by use of various means. A wide variety of means for acetone detection are known in the art, including those for detecting acetone from liquid solution (for example, blood and plasma analysis, urine testing) and those for detecting acetone from gas mixtures (for example, breath sampling, ambient gas monitoring). A broad assortment of different methodologies have been employed in acetone detection, monitoring; and analysis. These methodologies include those relying on, for example: color indicator, optical reflection, heat-of-combustion, electrical resistance, gas chromatography (GC), liquid chromatography (LC), photometry, colorimetry, ultraviolet spectrometry (UV), infra-red spectroscopy and spectrometry (IR), microwave spectroscopy, and mass spectrometry (MS) technologies.
These technologies, and thus the methodologies employing them, vary in their specificity: some detect a broad range of volatile organic compounds (VOCs); some detect either ketones (and ketoacids) alone or both ketones (and ketoacids) and aldehydes; and some detect acetone specifically.
In a first group of acetone detection methodologies, acetone is monitored by use of any of a variety of technologies that detect a broad range of VOCs, examples of which technologies include the following. Ambient gas monitors that detect a broad range of VOCs include, for example: the Dräger Polytron SE Ex detector, which employs a catalytic, heat-of-combustion “pellisitor” type sensor (catalog no. 68 09 760; 0.60 kilograms); and the Dräger Polytron IR Ex gas detector, which uses an infrared sensor (catalog no. 83 12 550; 1.9 kilograms) (both available from Dräger Sicherheitstechnik GmbH, Lübeck, Germany).
Fluid-solid interaction-based detection of VOCs relies upon gas or liquid adsorption onto a solid phase (optionally including a chemical derivatization reaction), followed by colorimetric or photometric detection of the adsorbed and/or derivatized compound(s). Such fluid-solid interaction technologies are discussed in British Patent No. 1082525, which discloses detection of organic compounds containing active or activated hydrogen atoms, wherein metal zeolites are used as the solid. VOC detection involving liquid adsorption onto solid and utilizing photometric detection is discussed in U.S. Pat. No. 4,882,499. This patent teaches a liquid detector utilizing fiber optics to detect changes in optical coefficient of reflection of liquid sample absorbed by capillary action; a hydrophobic fibrous or sintered matrix is used as the adsorptive solid for this purpose.
In another approach to VOC detection, gas that adsorbs onto a solid is sensed by electrical resistance/conductivity detection: this is described in U.S. Pat. No. 5,382,341. This patent describes a process of manufacturing smoke-detecting elements in which a bismuth oxide film is deposited onto a substrate layer, and, thereafter, is electrically connected to a means for measuring resistance. In this case, the solid may comprise bismuth oxides, Bi—Fe-oxides, Bi—V-oxides, or Bi—Mo-oxides. Similarly, German Patent No. 028062 describes a gas adsorption method in which the solid comprises an adsorbent layer of a semiconductor device.
A further methodology for VOC detection relies upon a gas phase chemical derivatization (halogenation) reaction to produce a detectable halogenated product. Examples of such gas phase derivatization methodologies for VOC detection are taught in U.S. Pat. No. 4,198,208 (describing reaction with chlorine and detection of chlorinated species) and in German Publication No. 4007375. All of the technologies in this first group are capable of, and taught for, detecting acetone, though non-specifically as a member of the class of VOCs.
A second group of methodologies used for acetone detection are those that detect ketones/ketoacids or both ketones/ketoacids and aldehydes generally. The technologies utilized in these methods depend on chemical derivatization of, for example, acetone, to produce a colored product. The colored product can then be visually inspected to obtain a qualitative result. Similarly, the colored product can be visually compared with a color standard chart to obtain a semi-quantitative reading. Alternatively, the degree of coloration can be quantitatively assessed by means of colorimetry or photometry. The most common derivatization reactions employed in these technologies are those based on: 1) reaction with salicylaldehyde; 2) reaction with hydrazine (or a phenylhydrazine, for example, 2,4-dinitrophenylhydrazine); and 3) reaction with nitroprusside. Many other chemical derivatization reactions are known, but are not commonly employed, for detection of aldehydes and ketones/ketoacids (including acetone), because those reactions use high temperatures or caustic, non-durable, or expensive reagents that make widespread use impractical.
In salicylaldehyde-based assays, the ketone or ketoacid is introduced to an alkaline solution containing salicylaldehyde, whereupon an orange or red derivative is produced. For example, both acetone and acetoacetate may be detected in urine by this route, as disclosed in U.S. Pat. No. 2,283,262.
In hydrazine-based and phenylhydrazine-based assays, aldehydes, ketones, and ketoacids are derivatized to form one or more hydrazone or phenylhydrazone compounds. For example, gas phase acetone absorption into liquid solution for chemical derivatization by this route is described: in U.S. Pat. No. 4,931,404, which discloses derivatization by reaction with a hydrazine- or phenylhydrazine-coupled cation exchange matrix, followed by colorimetric detection of the, for example, yellow, derivative; and in British Patent No. 2253910, which discloses derivatization by reaction with a hydrazine solution, followed by electrical resistance detection of the derivative.
In nitroprusside-based assays, aldehydes, ketones, and ketoacids are reacted with nitroprusside, that is a salt of nitroprussic acid (for example, sodium nitroprusside, that is sodium nitroferricyanide), to form a derivative(s) that, in the presence of an amine, forms a pink or purple complex. In some methods, the amine is present during the nitroprusside reaction for immediate coloration, while in others, an amine-containing solution is added later to develop the color. The nitroprusside reaction is the one most commonly used to detect acetone in the context of personal health monitoring, for example, in diabetes or in weight loss. Such nitroprusside methodology is typically found in one of three different formats: gas sampling tubes, fluid testing strips, and fluid testing tablets.
A variety of nitroprusside tube assay devices are commonly used. One of the most common is the Draeger tube (that is the Dräger tube), for example, the Draeger acetone detector tube (catalog no. DRAG CH22901 from SAFECO, Inc., Knoxville, Tenn.). The Draeger acetone detector tube can be used for breath analysis, but is mainly employed for ambient gas sampling in which a pump draws an air sample through the tube. A similar assay employs the Draeger Chip Measurement System (catalog no. 540-CMS from Safety First of Middleton, Wis.; 0.74 kilograms) which utilizes a “chip,” that is a planar, parallel array of Draeger tubes of capillary dimension. This hand-held device pumps a gas sample into the capillary tube(s), and the optics and electronics within the device perform colorimetry to convert the degree of coloration of the derivative within the tube into a quantitative digital signal. Similarly, methods for quantitatively monitoring acetone (and other ketones) in gas samples by photometric detection of the colored derivative, are taught in manufacturer information available with MSA acetone detector tubes (catalog no. 226620, available from Ben Meadows Co., a subsidiary of Lab Safety Supply Inc., PO Box 5277, Janesville, Wis. 53547). Also, U.S. Pat. No. 5,174,959 discloses a nitroprusside tube assay device containing two solid matrices: a nitroprusside-coupled matrix and an amine-coupled matrix. The device may be used with gas samples, in which case a solvent such as methanol is added, or with liquid samples such as urine.
Nitroprusside fluid testing strips and tablets are typically marketed as urine ketone test strips and tablets. Examples of such ketone test strip products are CHEMSTRIP K (produced by Roche Diagnostics Corp., Indianapolis, Ind.,) and KETOSTIX (produced by Bayer Corp., Diagnostics Division, Tarrytown, N.Y.). Exemplary ketone test tablets include the AMES ACETEST reagent tablets (catalog no. AM-2381, available from Analytical Scientific, Ltd., San Antonio, Tex.).
A third group of methodologies are acetone-specific. These acetone-specific detection methodologies utilize analytical devices and techniques. For example, acetone-specific detection methodologies include: gas chromatography detection; liquid chromatography detection using a micro-column, as disclosed in U.S. Pat. No. 6,063,283; and mass spectrometry detection, as described in U.S. Pat. No. 5,999,886, for use in the context of acetone vapor detection in semiconductor wafer processing chambers.
Specific detection of acetone in fluids by IR spectroscopy is disclosed in U.S. Pat. No. 5,355,425 (for liquids) and U.S. Pat. No. 4,587,427 (for gases). Specific detection of acetone in gases by FTIR spectrometry is disclosed in R T Kroutil et al., “Automated Detection of Acetone, Methyl Ethyl Ketone, and Sulfur Hexafluoride by Direct Analysis by Fourier-Transform Infrared Interferograms,” Applied Spectroscopy 48(6):724-32 (June 1994). Specific detection of acetone in gases by microwave spectroscopy is described in the abstract of Medical Technology Co-Operation Offer 131.C, entitled “Microwave Gas Spectroscopy for the Analysis of Exhaled Air,” from the Nizhny Novgorod Region Cooperation of the East-West Agency (OWA) of the Association for Innovation Research and Consultation mbH of the Innovation Consulting Institute (InnovationsBeratungsInstitut, Dusseldorf, Del.).
In addition, a number of acetone-specific detection methods employing hyphenated analytical techniques are also well known in the art. For example, a selection of such methods, including those relying on GC-HPLC, GC-FID (“Flame Ionization Detector”), GC-MS, GC-RGD (“Reduction Gas Detector”), and HPLC-UV techniques, are described in J D Reisman, Environmental Health Criteria for Acetone (Draft), Environmental Health Criteria No. 207, International Programme on Chemical Safety (INCHEM) (1998).
Thus, gas (into liquid) absorption with chemical reactions, gas (onto solid) adsorption with and without chemical reactions, liquid (onto solid) adsorption with and without chemical reactions, gas phase chemical reactions, solution phase chemical reactions, UV, IR, GC, LC, MS, and other technologies have all been used for acetone detection. However, all of these technologies have drawbacks.
For example, it is desirable, for environmental, health, and safety reasons, to select an acetone detection method that is specific for acetone. This is especially important in the area of subject self-monitoring, for example, for diabetes and for dieting. Thus, broad VOC detection technologies are less desirable for these purposes. While the acetone-specific methodologies currently in use are specific for acetone, they require bulky equipment that is not easily transportable and is relatively expensive to obtain and maintain, and they are impractical for use by individuals lacking medical or scientific training. It is desirable for acetone detection technology to be light-weight, readily transportable, low cost, and easy-to-use. These features are also especially important in the area of subject self-monitoring. Thus, the currently available acetone-specific detection technologies are less desirable for these purposes and reasons.
In contrast to these acetone-specific technologies, most of the currently available methodologies that detect ketones/ketoacids or both ketones/ketoacids and aldehydes generally are relatively inexpensive, light-weight, readily transportable, and easy-to-use. Yet, without the use of a secondary detection system, such as colorimetry or photometry, these tests produce only a qualitative or semi-quantitative result: a visualized color reading. Second, these tests are not specific for acetone: the presence of acetoacetate, other ketones, and aldehydes can result in a falsely intensified color reading. Finally, these tests are susceptible of producing false positive and false negative results; the former falsely indicate the presence of elevated acetone, the latter falsely indicate the absence of elevated acetone. For example, in the most commonly used assays (nitroprusside assays): false positive results often occur with subjects taking, for example, sulfhydryl drugs such as captopril, or when other ketones or aldehydes are present; and false negative results often occur when testing highly acidic samples, for example, samples of urine from subjects taking large doses of vitamin C (ascorbic acid). It is desirable for acetone detection technology to be reliable, to be specific for acetone, and to be capable of producing a directly quantitative result. These features are also especially important in the area of subject self-monitoring. Thus, the currently available ketone/ketoacid and aldehyde detection technologies are less desirable for these purposes.
Therefore, a need exists in the field of acetone detection for an acetone detection technology that is acetone-specific, light-weight, readily transportable, low cost, easy-to-use, reliable, and capable of producing a directly quantitative result. It would also be advantageous for such a technology to be capable of producing an electronic result, for example, a digital result (or in the case of a photonic-type computer or other instrument, capable of producing a photonic result). Affordable, disposable, specific, single-use devices for monitoring acetone levels in biological samples by, for example, a subject at home, are not readily available.
Other fields, such as the field of ethanol detection, utilize enzyme-based technologies. Enzyme-based technologies can be analyte-specific and can take the form of light-weight, readily transportable, low cost, easy to use, and quantitative devices. For example, in the field of ethanol detection in biological samples, gas phase ethanol detection has been performed by means of an enzyme-linked electrochemical sensor, using either alcohol oxidase (AOX) or primary alcohol dehydrogenase (ADH) as the enzyme. In one, exemplary ethanol detection system, a thick-film, screen-printed enzyme electrode using ADH/NAD+ immobilized in hydroxyethylcellulose is utilized for monitoring ethanol vapor. This ethanol-detecting enzyme electrode is activated by dipping it into buffer, the ethanol-containing sample is applied, and the resulting NADH produced by enzymatic action is monitored amperometrically at 650 mV (vs. Ag/AgCl). A similar electrode has been described for measuring ethanol vapor, wherein ADH is immobilized in reverse micelle media. The ethanol is partitioned into the aqueous phase where it is effectively concentrated in the medium where ADH can act upon it. These technologies are readily adaptable to easily transportable, low-cost detectors that can be used to monitor or self-monitor breath ethanol at home, at work, and in other environments remote from clinics and testing laboratories. However, these devices are not designed for, and the enzymes utilized (for example, primary alcohol dehydrogenase) are not capable of, acetone detection. Thus, if it could be devised, an enzyme-based technology might be able to offer many of the benefits needed in the field of acetone detection.
Nevertheless, all of the above-described acetone detection methods and devices rely on non-enzymatic technologies. Enzymatic measurement of acetone in environmental and biological samples has not been described to date. Thus, it appears that, in addition to the need for an acetone detection technology having the advantageous features described above, the field of acetone detection is also lacking the use of, and thus the benefits of, enzyme-based technologies.